Biochemistry 2007, 46, 291-305
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Thermodynamic Investigation into the Mechanisms of Proton-Coupled Electron Transfer Events in Heme Protein Maquettes† Amit R. Reddi, Charles J. Reedy, Steven Mui, and Brian R. Gibney* Department of Chemistry, Columbia UniVersity, 3000 Broadway, MC 3121, New York, New York 10027 ReceiVed August 8, 2006; ReVised Manuscript ReceiVed NoVember 1, 2006
ABSTRACT: To study the engineering requirements for proton pumping in energy-converting enzymes such as cytochrome c oxidase, the thermodynamics and mechanisms of proton-coupled electron transfer in two designed heme proteins are elucidated. Both heme protein maquettes chosen, heme b-[H10A24]2 and heme b-[∆7-His]2, are four-R-helix bundles that display pH-dependent heme midpoint potential modulations, or redox-Bohr effects. Detailed equilibrium binding studies of ferric and ferrous heme b with these maquettes allow the individual contributions of heme-protein association, iron-histidine ligation, and heme-protein electrostatics to be elucidated. These data demonstrate that the larger, less well-structured [H10A24]2 binds heme b in both oxidation states tighter than the smaller and more well-structured [∆7His]2 due to a stronger porphyrin-protein hydrophobic interaction. The 66 mV (1.5 kcal/mol) difference in their heme reduction potentials observed at pH 8.0 is due mostly to stabilization of ferrous heme in [H10A24]2 relative to [∆7-His]2. The data indicate that porphyrin-protein hydrophobic interactions and heme iron coordination are responsible for the Kd value of 37 nM for the heme b-[∆7-His]2 scaffold, while the affinity of heme b for [H10A24]2 is 20-fold tighter due to a combination of porphyrin-protein hydrophobic interactions, iron coordination, and electrostatic effects. The data also illustrate that the contribution of bis-His coordination to ferrous heme protein affinity is limited, 6.6), MES (5.5 < pH < 6.5), and citrate (pH < 5.0). pH Dependence of the Peptide-Bound Heme b Reduction Potential As Determined by Heme Redox Potentiometry. The reduction potential as a function of pH for [H10A24]2 shows two distinct proton-coupled electron transfer events with 60 mV per pH slopes indicative of a 1H+/1e- coupled event (20). The data between pH 8.0 and 3.0 are best fit to an equilibrium model in which there is a one-proton/oneelectron electrochemical event.
RT 1 + 10-pH+pKa ln nF 1 + 10-pH+pKaox
red
Emeas ) Eacid -
(1)
where Emeas is the measured reduction potential at a given pH, Eacid is the limiting reduction potential at low pH corresponding to the Fe(III)/Fe(II) couple of the fully protonated heme protein, and pKared (7.0) and pKaox (4.2) are the pKa values in the reduced and oxidized states, respectively, of the residue that leads to the pH-dependent reduction potential. IR spectroscopy has been used to assign the residue responsible for the redox-Bohr effect to a glutamic acid (20). The heme reduction potential as a function of pH for heme b-[∆7-His]2 displays a 120 mV per pH slope indicative of a 2H+/1e- coupled event. Thus, the data can be fit to a proton-coupled electron transfer model in which there is one two-proton/one-electron electrochemical event.
RT 1 + 10-2pH+2pKa ln Emeas ) Eacid nF 1 + 10-2pH+2pKaox
red
(2)
Alternatively, the data may also be adequately described by a standard Henderson-Hasselbalch equation for the protonation of the two histidine residues and the concomitant dissociation of the heme group in the reduced state (37).
(
Emeas ) Eacid - ∆E
10
1 -pH+pKaeff-red
+ +1 1 -pH+pKaeff-red
10
+1
)
(3)
where the midpoint reduction potential measured at any pH, Emeas, is a function of the midpoint potential at acidic pH values, Eacid, the change in midpoint reduction potential due to deprotonation, ∆E, the solution pH value, and the acid dissociation constant of the histidine ligands in the presence of metal, the effective pKa value or pKaeff-red. Potentiometric pH Titrations in Heme Affinity Studies. Potentiometric pH titrations were performed manually using
Proton-Coupled Electron Transfer Mechanisms
Biochemistry, Vol. 46, No. 1, 2007 293
an anaerobic 1.0 cm path length cuvette fitted with a pH electrode under a stream of nitrogen gas. The pH of 3.0 µM heme protein samples in a combination buffer (20 mM HEPES, 20 mM MES, 20 mM citrate, and 100 mM KCl) was adjusted via addition of microliter aliquots of 6 N HCl. Between each addition, the samples were allowed to equilibrate for up to 20 min prior to measurement of the heme protein absorption by UV-vis spectroscopy. For the ferrous heme protein samples, sodium dithionite was added to ensure complete reduction prior to the start of the titration. The pH dependence of the Soret band absorbance at λmax was fit to an equation for the cooperative protonation of the two heme ligating histidine residues, as shown for the oxidized state.
1 Absmeas ) Abs0 + ∆Abs -2pH+2pKaeff-ox 10 +1
(4)
where the absorbance measured at any pH, Absmeas, is a function of the initial absorbance, Abs0, the change in absorbance due to protonation, ∆Abs, the solution pH value, the number of protons involved in the process, two, and the acid dissociation constant of the ligands in the presence of metal, the effective pKa value or pKaeff-ox. An analogous equation for the reduced state provides the reduced state effective pKaeff-red value of the histidine ligands. Ferric and Ferrous Heme b Conditional Dissociation Constants in Heme Affinity Studies. Ferric and ferrous heme b dissociation constants were determined over the pH range of 1.6-8.0 using previously reported titration methods (29). The buffers employed at various solution pHs were as follows: 20 mM KPi and 100 mM KCl at pH 8.0, 20 mM HEPES and 100 mM KCl at pH >6.6, 20 mM MES and 100 mM KCl between pH 5.5 and 6.5, and 20 mM citrate and 100 mM KCl at pH 6.0) to overnight (pH
